Introduction

Low back pain (LBP) is a worldwide leading cause of disability. This preclinical study evaluated the safety of a combined advanced therapy medicinal product developed during the European iPSpine project (#825925) consisting of mesendoderm progenitor cells (MEPC), derived from human induced pluripotent stem cells, in combination with a synthetic poly(N-isopropylacrylamide) hydrogel (NPgel) in an ovine intervertebral disc degeneration (IDD) model.

Introduction

To develop an international guideline (AOGO) about use of osteobiologics in Anterior Cervical Discectomy and Fusion (ACDF) for treating degenerative spine conditions.

Introduction

Intraoperative navigation systems for lumbar spine surgery allow to perform preoperative planning and visualize the real-time trajectory of pedicle screws. The aim of this study was to evaluate the deviation from preoperative planning and the correlations between screw deviation and accuracy.

Introduction

Intervertebral disc degeneration (IDD) is a progressive process affecting all disc tissues, namely the nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplates (CEPs). Several cell-based therapies have been proposed to replenish the disc cell population and promote tissue regeneration. However, cell-free therapeutics have been increasingly explored due to potentially higher advantages and cost-effectiveness compared to cell transplantation. Recently, extracellular vesicles (EVs) isolated from healthy Tie2⁺-NP cells (NPCs) have shown promising regenerative outcomes on degenerative NPCs (dNPCs). The aim of this study was to assess the effect of such EVs on all disc cell types, including AF cells (AFCs) and CEP cells (CEPCs), compared to EVs isolated from bone-marrow derived mesenchymal stromal cells (BM-MSCs).

Introduction

Osteoarthritis (OA) is a predominant chronic degenerative disease exerting a deep impact on quality of life and healthcare systems. Recent evidences suggest that pyroptosis, a programmed cell death characterized by inflammatory cytokine release, may play a significant role in modulating OA pain. The aim of the study is to investigate the potential role of extracellular vesicles derived from umbilical cord Wharton's jelly (WJ-MSC EVs) in the attenuation of the pyroptotic process on human chondrocytes (hOAC) pre-treated with synovial fluid in a 3D in vitro model.

Chronic low back pain (CLBP) is the most common cause of disability worldwide, and lumbar spine fusion (LSF) is often chosen to treat pain caused by advanced degenerative disease when clinical treatment failed certain cases, the post-surgical outcomes are not what was expected. Several studies highlight how important are. In psychological variables during the postoperative spine surgery period. The aim of this study is to assess the role of preoperative depression on postoperative clinical outcomes. We included patients who underwent LSF since December 2021. Preoperative depression was assessed administering Beck Depression Inventory questionnaire (BDI). And pain and disability were evaluated at 1, 3, and 6 months, administering respectively Visual Analogic Scale (VAS) and Oswestry Disability Index (ODI). As statistical analysis Mann-Whitney test was performed. We included 46 patients, 20 female (43,5%) and 26 male (56,5%) with an average age of 64,2. The population was divided in two groups, fixing the BDI cut-off point at 10. Patients with BDI < 10 points (N=28) had normal mental health status, instead patients with BDI > 10 points (N=16) had depressive disorders. At 3 months patients with healthy mental status reported statistically significant reduction of pain (U = 372,5, p = .006) and improvement of disability but without statistical significancy (U = 318, p = 0,137). At 6 months patients without psychological disease reported statistically significant reduction of pain (U = 342, p = 0,039) and disability (U = 372,5, p = 0,006).

This study demonstrates the correlation between pre-existing depressive state and poorer clinical outcomes after spine surgery. These results are consistent with the literature. Therefore, during the surgical decision making it is crucial to take psychological variables into account in order to predict the results after surgery and inform patients on the potential influence of mental status.

Low back pain (LBP) is the main cause of disability worldwide and is primarily triggered by intervertebral disc degeneration (IDD). Although several treatment options exist, no therapeutic tool has demonstrated to halt the progressive course of IDD. Therefore, several clinical trials are being conducted to investigate different strategies to regenerate the intervertebral disc, with numerous studies not reaching completion nor being published. The aim of this study was to analyze the publication status of clinical trials on novel regenerative treatments for IDD by funding source and identify critical obstacles preventing their conclusion.

Prospective clinical trials investigating regenerative treatments for IDD and registered on ClinicalTrials.gov were included. Primary outcomes were publication status and investigational treatment funding. Fisher's exact test was utilized to test the association for categorical variables between groups.

25 clinical trials were identified. Among these, only 6 (24%) have been published. The most common source of funding was university (52%), followed by industry (36%) and private companies (12%). Investigational treatments included autologous (56%) or allogeneic (12%) products alone or in combination with a carrier or delivery system (32%). The latter were more likely utilized in industry or privately funded studies (Fig. 1, p=0.0112). No significant difference was found in terms of funding regarding the publication status of included trials (Table 1, p=0.9104).

Most clinical trials investigating regenerative approaches for the treatment of IDD were never completed nor published. This is likely due to multiple factors, including difficult enrollment, high dropout rate, and publication bias³. More accurate design and technical support from stakeholders and clinical research organization (CROs) may likely increase the quality of future clinical trials in the field.

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Despite promising results in attempting intervertebral disc regeneration, intradiscal cell transplantation is affected by several drawbacks, including poor viability in the harsh disc environment, low cost-effectiveness, and immunogenic/tumorigenic concerns. Recently, the development of cell-free approaches is gaining increasing interest in the field, with a particular regard towards extracellular vesicles (EVs). Nucleus pulposus cell (NPC) progenitors characterized by Tie2 expression have shown a higher chondrogenic differentiation potential compared to MSCs. The aim of this study was to investigate the putative regenerative effects of EVs isolated from Tie2-overexpressing NPC progenitors on degenerative NPCs.

NPCs were isolated from young donors and underwent an optimized culture protocol to maximize Tie2 expression (NPCs^Tie2+) or a standard protocol (NPCs^STD). Following EV characterization, NPC isolated from patients affected by intervertebral disc degeneration (IDD) were treated with either NPCs^Tie2+-EVs or NPCs^STD-EVs. Cell proliferation and viability were assessed with the CCK-8 assay. Cell apoptosis and necrosis were evaluated with the Annexin V/PI assay. Cell senescence was investigated with b-galactosidase staining. EV uptake was assessed with PKH26 staining of EVs under confocal microscopy.

Treatment with EVs isolated from young NPC donors significantly increased degenerative NPC viability, especially in samples treated with NPCs^Tie2+-EVs. Likewise, NPCs^Tie2+-EVs significantly reduced cell senescence and did not show to exert necrotic nor apoptotic effects on recipient cells. Furthermore, EV uptake was successfully observed in all treated cells.

NPCs^Tie2+-EVs demonstrated to significantly enhance degenerative NPC viability, senescence and apoptosis. The use of committed progenitors naturally residing the in the nucleus pulposus may optimize EV regenerative properties and constitute the basis for a new therapy for IDD.

Low back pain (LBP) is a worldwide leading cause of disability. Treatment of intervertebral disc (IVD) with stem cells has been used on degenerate discs (IDD), cause of around 40% of LBP cases. Despite pain reduction, clinical studies' follow-up have not shown a structural IVD improvement. A valid alternative may be the use of notocordal cells (NC) or their precursors. Mesendoderm progenitor cells (MEPC) have the ability to replicate and differentiate toward NC. In this preliminary study we evaluated in a preclinical IDD model the viability and NC differentiation of MEPC derived from induced pluripotent stem cells (iPSC).

MEPC derived from iPSC were developed during the iPSpine project (# 825925), thawed, plated for 24h on laminin and labeled with PKH26.

Two adult sheep were subjected to nucleotomy of five lumbar discs for the induction of IDD. After 5 weeks, 3 degenerated discs were treated with MEPC at 3 different doses (low, medium and high). One sheep was sacrificed after 7 days and one after 30 days. Clinical parameters were collected to evaluate the safety of treatment. Discs were analysed using histological techniques. Survival (PKH26), proliferation (PCNA), notocordal cell differentiation (Brachyury, Cytokeratin 8/18/19, Sox9, Foxa2) and endodermal differentiation (Sox17) were evaluated.

At 7 days from treatment, both sheep lost about 20% of body weight. Only in discs treated with the highest dose PKH26 stained cells were alive up to 30 days. These cells turn out to be: proliferating (PCNA); positive for Brachyury, cytokeratin 8/18/19 and Foxa2; positive for SOX17 in a small percentage.

This preliminary study shows that MEPC, derived from iPSC and injected into ovine discs degenerated by nucleotomy, are able to survive up to 30 days and differentiate within the disc predominantly towards the notocordal phenotype.

Infections are among the most diffused complications of the implantation of medical devices. In orthopedics, they pose severe societal and economic burden and interfere with the capability of the implants to integrate in the host bone, significantly increasing failure risk. Infection is particularly severe in the case of comorbidities and especially bone tumors, since oncologic patients are fragile, have higher infection rate and impaired osteoregenerative capabilities. For this reason, prevention of infection is to be preferred over treatment.

This is even more important in the case of spine surgery, since spine is among the main site for tumor metastases and because incidence of post operative surgical-site infections is significant (up to 15-20%) and surgical options are limited by the need of avoiding damaging the spinal cord.

Functionalization of the implant surfaces, so as to address infection and, possibly, co- adjuvate anti-tumor treatments, appears as a breakthrough innovation. Unmet clinical needs in infection and tumors is presented, with a specific focus on the spine, then, new perspectives are highlighted for their treatment.

Invertebral disc degeneration (IDD) is a degenerative disease involving a variety of musculoskeletal and spinal disorders such as lower back pain (LBP). Secretome derived from mesenchymal stem cells (MSCs) have exerted beneficial effect on tissue regeneration. In this study, the goal was to investigate the paracrine and the anti-inflammatory effects of secretome from interleukin IL1β preconditioned Bone Marrow MSCs (BMSCs) on human nucleus pulposus cells (hNPCs) in a 3D in vitro model.

Secretome was collected from BMSCs (BMSCs-sec) after preconditioning with 10 ng/mL IL1β. hNPCs were isolated from surgical specimens, culture expanded in vitro, encapsulated in alginate beads and treated with: growth medium; IL1β 10 ng/mL; IL1β 10 ng/mL for 24 hours and then BMSCs-sec. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess assay) and ROS quantification (Immunofluorescence) iii) glycosaminoglycan (GAG) amount (DMBB) and iv) gene expression levels of extracellular matrix (ECM) components and inflammatory mediators (qPCR). One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D.

In vitro tests showed an enhancement of hNPCs proliferation after treatment with BMSCs-sec (p ≤ 0.05) compared to IL1β group. After 24 hours, the percentage of dead cells was higher in IL1β treated hNPCs compared to control group and decreased significantly in combined IL1β and BMSCs-sec sample group (p ≤ 0.01). Nitrite and ROS production were significantly mitigated and GAGs content was improved by preconditioned BMSCs-sec (p ≤ 0.05). Furthermore, gene expression levels were modulated by BMSCs-sec treatment compared to controls.

Our results supported the potential use of BMSCs' secretome as a cell-free strategy for IDD, overcoming the side effects of cell-therapy. Moreover, secretome derived from IL1β preconditioned BMSCs was able to reduce hNPCs death, attenuate ECM degradation and oxidative stress counteracting IDD progression.

Acknowledgements: Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects.

The use of intraoperative navigation and robotic surgery for minimally invasive lumbar fusion has been increasing over the past decade. The aim of this study is to evaluate postoperative clinical outcomes, intraoperative parameters, and accuracy of pedicle screw insertion guided by intraoperative navigation in patients undergoing lumbar interbody fusion for spondylolisthesis. Patients who underwent posterior lumbar fusion interbody using intraoperative 3D navigation since December 2021 were included. Visual Analogue Scale (VAS), Oswestry Disability Index (ODI), and Short Form Health Survey-36 (SF-36) were assessed preoperatively and postoperatively at 1, 3, and 6 months. Screw placement accuracy, measured by Gertzbein and Robbins classification, and facet joint infringement, measured by Yson classification, were assessed by intraoperative Cone Beam CT scans performed at the end of instrumentation. Finally, operation time, intraoperative blood loss, hospital stay, and screw insertion time were evaluated. This study involved 50 patients with a mean age of 63.7 years. VAS decreased from 65.8±23 to 20±22 (p<.01). ODI decreased from 35.4%±15 to 11.8%±14 (p<.01). An increase of SF-36 from 51.5±14 to 76±13 (p<.01) was demonstrated. The accuracy of “perfect” and “clinically acceptable” pedicle screw fixation was 89.5% and 98.4%, respectively. Regarding facet violation, 96.8% of the screws were at grade 0. Finally, the average screw insertion time was 4.3±2 min, hospital stay was 4.2±0.8 days, operation time was 205±53 min, and blood loss was 169±107 ml. Finally, a statistically significant correlation of operation time with hospital stay, blood loss and placement time per screw was found. We demonstrated excellent results for accuracy of pedicle screw fixation and violation of facet joints. VAS, ODI and SF-36 showed statistically significant improvements from the control at one month after surgery.

Navigation with intraoperative 3D images represents an effective system to improve operative performance in the surgical treatment of spondylolisthesis.

This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation.

hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 5, 10 and 25 ng/mL (n=4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and −3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by RT-PCR. MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin).

Irisin increased hNPC proliferation (p<0.001), metabolic activity (p<0.05), GAG content (p<0.01), as well as COL2 (p<0.01), aggrecan (p<0.05), TIMP-1 and −3 (p<0.01) gene expression, while decreasing MMP-13 (p<0.05) and IL-1β (p<0.001) mRNA levels. r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p<0.001) and a decrease of IL-1β (p<0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin increased hNPC metabolic activity (p<0.001), COL2 gene expression (p<0.05) and decreased IL-1β (p<0.05) and ADAMTS-5 levels (p<0.01).

Irisin stimulates hNPC proliferation, metabolic activity, and anabolism by reducing IL-1β and catabolic enzyme expression while promoting matrix synthesis.

The purpose of this study was to evaluate the beneficial effects of r-Irisin (IR) on human primary tenocytes (hTCs) in vitro. Indeed, Irisin is secreted from muscles in response to exercise and mediates many beneficial effects on tissues and organs.

Tissue samples (n=3) were analyzed by histology and immunohistochemistry for αVβ5 receptor. hTCs isolated, culture expanded were treated with: 1) RPMI medium as control; 2) IR at different concentrations; 3) IL-1β; 4) pre-treated with IL-1β for 24 h and then co-treated with IR; 5) pre-treated with IR for 24 h and then co-treated with IL-1β. We evaluated: cell metabolic activity (MTT); cell proliferation (trypan blue staining and PicoGreen); nitrite concentration (Griess). The analysis were performed in triplicate for each donor and each experiment was repeated at least three times. Data were expressed as mean ± S.D. One-way ANOVA analysis was used to compare the groups under exam.

We found the presence of the αVβ5 receptor on hTCs plasma membrane supporting the potential interaction with irisin. Cell proliferation was significantly increased with IR at 5, 10 and 25 ng/mL. IR 25 ng/mL after IL1β pre-treatment was able to counteract the increase of nitrite production (p < 0.001) compared to the inflamed hTCs (p < 0.01; p < 0.0001), as well as IR at 10 and 25 ng/ml showed a protective role from oxidative damage. We observed a significant increase in cell metabolic viability in culture under IR at 5 and 25 ng/mL (p < 0.001; p < 0.05) in the pre-treated IR groups, whereas IR showed anti-inflammatory effects at the highest concentration of r-Irisin (p < 0.05).

This is the first study reporting the capability of irisin to attenuate tendinopathy in vitro by acting on acute inflamed tenocytes. Our results confirmed and highlighted the potential cross-talk mechanism between muscle and tendon.

The aim of this study was to investigate the regenerative effects of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) derived exosomes (WJ-Exos) on human nucleus pulposus cells (hNPCs) in an in vitro 3D model.

WJ-Exos were isolated by tangent flow filtration of WJ-MSCs conditioned media and characterized by TEM, WB for markers expression and quantified with NTA. WJ-Exos PKH26-labeled uptake in hNPCs was detected by confocal microscopy. hNPCs, isolated from surgical specimens (n=4), culture expanded in vitro and encapsulated in alginate beads, were pre-treated with IL1β (10 ng/ml) for 24 hours, then with WJ-Exos at 10, 50 and 100 µg/ml. Cells with growth medium were used as control. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess) iii) glycosaminoglycan (GAG) amount (DMBB), iv) histological staining for extracellular matrix (ECM) analysis and v) gene expression levels of catabolic and anabolic genes (qPCR). The investigations were performed in triplicate for each donor. One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D.

A dose dependent increase in hNPCs proliferation was noticed at all exos concentrations under study. Cell death decreased significantly in WJ-Exos 50 µg/ml samples (p ≤ 0,05) compared to IL1β treated hNPCs. Nitrite production was significantly attenuated at 10µg/ml of WJ-Exos (p ≤ 0,01). GAG content and histological analysis showed a difference in ECM synthesis between treated and untreated hNPCs (p ≤ 0,05). Catabolic and inflammatory markers were modulated by WJ-Exos at 100 µg/ml concentration (p ≤ 0,05) whereas 10 µg/ml group increased anabolic gene expression levels (p ≤ 0,05).

These findings offer new opportunities for the potential use of exosomes as an attractive alternative cell-free strategy of IDD. WJ-MSC exosomes ameliorate hNPCs growth and viability, attenuate ECM degradation and oxidative stress-related IDD progression after IL1β stimulation.

Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects.

Intraneural electrodes can be harnessed to control neural prosthetic devices in human amputees. However, in chronic implants we witness a gradual loss of device functionality and electrode isolation due to a nonspecific inflammatory response to the implanted material, called foreign body reaction (FBR). FBR may eventually lead to a fibrous encapsulation of the electrode surface. Poly(ethylene glycol) (PEG) is one of the most common low-fouling materials used to coat and protect electrode surfaces. Yet, PEG can easily undergo encapsulation and oxidative damage in long-term in vivo applications. Poly(sulfobetaine methacrylate) - poly(SBMA) - zwitterionic hydrogels may represent more promising alternatives to minimize the FBR due to their ultra-low fouling features. Here, we tested and compared the poly(SBMA) zwitterionic hydrogel coating with the PEG coating in reducing adhesion and activation of pro-inflammatory and pro-fibrotic cells to polyimide surfaces, which are early hallmarks of FBR. We aimed to coat polyimide surfaces with a hydrogel thin film and analysed the release of a model drug from the hydrogel.

We performed hydrogel synthesis, mechanical characterization and biocompatibility analysis. Cell adhesion, viability and morphology of human myofibroblasts cultured on PEG- and hydrogel-coated surfaces were evaluated through confocal microscopy-based high-content analysis (HCA). Reduced activation of pro-inflammatory human macrophages cultured on hydrogels was assessed as well as the hydrogel drug release profile.

Because of its high hydration, biocompatibility, low stiffness and ultra-low fouling characteristics the hydrogel enabled lower adhesion and activation of pro-inflammatory and pro-fibrotic cells vs. polystyrene controls, and showed a long-term release of the anti-fibrotic drug Everolimus. Furthermore, a polyimide surface was successfully coated with a hydrogel thin film.

Our soft zwitterionic hydrogel could outperform PEG as more suitable coating material of neural electrodes for mitigating the FBR. Such poly(SBMA)-based biomaterial could also be envisioned as long-term delivery system for a sustained release of anti-inflammatory and anti-fibrotic drugs in vivo.

With the coronavirus disease 2019 (COVID-19) pandemic, remote working has been ubiquitously implemented to reduce disease transmission via minimization of in-person interactions. Low back pain (LBP) is the first cause of disability worldwide and is frequently reported by workers with sedentary occupations. This cross-sectional study aimed to assess the role of remote working in a population of adults affected by LBP through an online questionnaire.

We enrolled 136 teleworkers affected by LBP. A total of 101 responses were received and 93 suitable questionnaires were included in the final analysis. Demographic data, remote working features and tasks, and LBP burden were analysed. The psychological burden of remote working was evaluated with the World Health Organization Five Well-Being Index (WHO-5) and the Patient Health Questionnaire-2 (PHQ-2). LBP severity was evaluated using a visual analog scale (VAS). LBP-related disability was assessed using the Oswestry Disability Index (ODI). The effect of LBP on working capacity was examined with the Occupational Role Questionnaire (ORQ). Independent risk factors related to LBP worsening were identified using a multivariate logistic regression model.

LBP severity was significantly higher compared to previous in-person working (p<0.0001) as well as average weekly work hours (p<0.001). Furthermore, the risk of LBP deterioration was associated with being divorced (OR: 4.28, 95% CI: 1.27-14.47; p=0.019) or living with others (OR: 0.24, 95% CI: 0.07-0.81; p=0.021), higher ill-being (OR: 0.91, 95% CI: 0.83-0.99; p=0.035) and depression scores (OR: 1.38, 95% CI: 1.00-1.91; p=0.048), as well as having reported unchanged (OR: 0.22, 95% CI: 0.08-0.65; p=0.006) or decreased job satisfaction (OR: 0.16, 95% CI: 0.05-0.54; p=0.003) and increased stress levels (OR: 3.00, 95% CI: 1.04-8.65; p=0.042).

These findings highlight key factors to consider for improving remote workers’ physical and mental wellbeing and decrease their LBP burden.

Intervertebral disc degeneration (IDD) affects more than 80% of the population all over the world. Current strategies for the treatment of IDD are based on conservative or surgical procedures with the aim of relieving pain. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy in recent decades, but studies showed that the particularly hostile microenvironment in the intervertebral disc (IVD) can compromise cells survival rate. The use of exosomes, extracellular vesicles released by various cell types, possess considerable economic advantages including low immunogenicity and toxicity. Exosomes allow intercellular communication by conveying functional proteins, RNA, miRNA and lipids between cells. The purpose of this study is to assess the therapeutic effects of exosomes derived from Wharton Jelly mesenchymal stromal cells (WJ-MSC) on human nucleuspulposus cells (hNPC) in an in vitro 3D culture model.

Exosomes (exos) were isolated by tangential flow filtration of WJ-MSC conditioned media and characterized by: quantification with BCA test; morphological observation with TEM, surface marker expression by WB and size evaluation by NTA. Confocal microscopy has been used to identify exosomes marked with PKH26 and monitor fusion and/or incorporation in hNPC. hNPC were isolated from waste surgical material from patients undergoing discectomy (n = 5), expanded, encapsulated in alginate beads and treated with: culture medium (control group); WJ-MSC exos (WJ-exos) at different concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml).

They were then analysed for: cell proliferation (Trypan Blu); viability (Live/Dead Assay); quantification of nitrites (Griess) and glycosaminoglycans, GAG (DMBB). The hNPC in alginate beads treated for 7 days were included in paraffin and histologically analysed to determine the presence of extracellular matrix (ECM) components. Finally, the expression levels of catabolic and anabolic genes were evaluated through real-time polymerase chain reaction (qPCR).

All concentrations of WJ-exos under exam were capable to induce a significant increase in cell proliferation after 10 and 14 days of treatment (p < 0.01 and p < 0.001, respectively). Live/Dead assay showed a decrease in cell death at 50 μg/ml of WJ-exos (p < 0.05). While cellular oxidative stress indicator, nitrite production, was reduced in a dose-dependent way and statistically significant only with 100 μg/ml of WJ-exos (p < 0.05). WJ-exos at 10 and 100 μg/ml induced a significant increase in GAG content (p < 0.05; p < 0.01, respectively) confirmed by Alcian Blu staining. Exos derived from WJ-MSC modulated gene expression levels by increasing expression of ACAN and SOX-9 genes and reducing significantly of IL-6, MMP-1, MMP-13 and ADAMTS-5 levels (p < 0.05; p < 0.01) compared to the control group.

Our results supported the potential use of exosomes from WJ-MSC for the treatment of IDD. Exosomes improved hNPC growth, attenuated ECM degradation and reduced oxidative stress and inflammation. This study offers a new scenario in IVD regeneration, promoting the potential use of extracellular vesicles as an alternative strategy to cell therapy.

Low back pain (LBP) is the leading cause of disability worldwide. Recently, treatment of the intervertebral disc (IVD) with stem cells has been used for the treatment of degenerate discs (IDD) which cause at least the 40% of LBP cases. Despite pain reduction, follow-up in clinical studies have not shown an improvement in the structural integrity of IVD. A valid alternative could be the use of progenitor disc cells (notocordal cells, NC) or of their precursors. Mesendoderm progenitor cells (MEPC) have the ability to replicate and differentiate toward NC. In this preliminary study we evaluated in a preclinical large animal IDD model the viability and NC differentiation of MEPC derived from induced pluripotent stem cells (iPSC).

MEPC, derived from iPSC and developed during the iPSpine project (# 825925), were thawed and plated on laminin for 24h and labeled with PKH26.

Two adult sheep were subjected to nucleotomy of five lumbar discs for the induction of IDD. After 5 weeks, 3 of the 5 degenerate discs were treated with MEPC at 3 different doses (low, medium and high). One sheep was sacrificed after 7 days and the other after 30 days from the treatment injection procedure. Clinical parameters were collected to evaluate the safety of treatment. Discs were paraffin embedded and analysed using histological techniques. Survival (PKH26), proliferation (PCNA), notocordal cell differentiation (Brachyury, Cytokeratin 8/18/19, Sox9, Foxa2) and endodermal differentiation (Sox17) were evaluated.

After the injection of the cells, both sheep lost about 20% of body weight. The analysis showed that only in discs treated with the highest dose the PKH26 stained cells resulted alive after 30 days from the procedure. These cells turn out to be:

This preliminary study shows that MEPC, derived from iPSC and injected into ovine discs degenerated by nucleotomy, are able to survive 30 days from treatment and differentiate within the disc predominantly towards the notocordal phenotype.

Introduction:

Exercise has showed to reduce pain and improve function in patients with discogenic low back pain (LBP). Although there is currently no biologic evidence that the intervertebral disc (IVD) can respond to physical exercise in humans, a recent study has shown that chronic running exercise is associated with increased IVD hydration and hypertrophy1. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types, including chondrocytes2. This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs). Our hypothesis is that irisin may improve hNPCs metabolism and proliferation.